Bacillus anthracis is a soil-borne, Gram-positive endospore-forming bacterium and the
causative agent of anthrax. It is enzootic in Pafuri, Kruger National Park in South Africa. The bacterium
is amplified in a wild ungulate host, which then becomes a source of infection to the next host upon
its death. The exact mechanisms involving the onset (index case) and termination of an outbreak
are poorly understood, in part due to a paucity of information about the soil-based component of
the bacterium’s lifecycle. In this study, we present the unique isolation of a dsDNA bacteriophage
from a wildebeest carcass site suspected of having succumbed to anthrax. The aggressively lytic
bacteriophage hampered the initial isolation of B. anthracis from samples collected at the carcass site.
Classic bacteriologic methods were used to test the isolated phage on B. anthracis under di erent
conditions to simulate deteriorating carcass conditions. Whole genome sequencing was employed to
determine the relationship between the bacterium isolated on site and the bacteriophage-dubbed
Bacillus phage Crookii. The 154,012 bp phage belongs to Myoviridae and groups closely with another
African anthrax carcass-associated Bacillus phage WPh. Bacillus phage Crookii was lytic against
B. cereus sensu lato group members but demonstrated a greater a nity for encapsulated B. anthracis at
lower concentrations (<1 108 pfu) of bacteriophage. The unusual isolation of this bacteriophage
demonstrates the phage’s role in decreasing the inoculum in the environment and impact on the life
cycle of B. anthracis at a carcass site.
Table S1. To determine the Bacillus phage Crookii e ects on Bacillus anthracis in decomposing blood,
Romanowsky-Giemsa stained smears were visually appraised microscopically at three time points. The bacterial
counts (vegetative cells versus endospores) were enumerated under di erent conditions (standard incubation,
carbon dioxide incubation, sodium bicarbonate content and bacteriophage type) for comparison. Counts represent
cells/spores per 100 L of blood. Table S2. General features of the genome sequence of Bacillus anthracis DS201579
and Bacillus phage Crookii. Table S3. Five prophages of Bacillus anthracis DS201579 in the chromosome identified
using PHASTER. Table S4. De novo assembly of unmapped reads collected from read mapping analysis of Bacillus
anthracis DS201579 using B. anthracis Ames Ancestor as a reference genome (GenBank accession: NC_007530.2;
NC_007322.2 and NC_007323,2). Table S5: RAST annotation of contig sequences of the Bacillus phage Crookii
genome. Figure S1. Transmission electron micrographs of bacteriophage Bacillus phage Crookii, isolated from
an anthrax carcass site in Kruger National Park, South Africa. It was negatively stained with 2% uranyl acetate.
Bacillus phage Crookii has a Myoviridae morphology with a thickened contractile tail in relation to the head.
The head is ~80 nm with a tail measuring ~205 nm. Figure S2. The CGView comparative tool map of Bacillus
phage Crookii genome sequence to Bacillus phage WPh. Circles display from inside to outside: genomic position
in kilobases, GC skew (purple and green), GC content (black). Blast 1 results show the sequence homolog between
Bacillus phage Crookii and phage WPh (blast 2). Open reading frames are indicated as pink from the outside.